Purification Of Antigen Research Articles

Extraction, isolation, and concentration of calprotectin antigen (S100A8/S100A9) from granulocytes.

BackgroundCalprotectin is a promising biomarker for granulocyte activation. It is mainly measured in faeces as a marker for inflammatory bowel disease. A limitation is that there is no widely accepted calibrator.AimTo establish a method for purification of calprotectin from human granulocytes that is easily reproducible, reliable, and could contribute to a better agreement between different calprotectin methods.Methods and resultsCalprotectin was purified from granulocyte extracts using ion‐exchange chromatography. The granulocytes were separated from blood bags. The purity was analysed by analysing pixel density of a picture of the sodium dodecyl sulfate polyacrylamide gel electrophoresis and by size exclusion chromatography. The calprotectin concentration of the pure antigen solution was determined using Biuret method. The purity was >95% for 3 preparations, and their concentrations were 1079, 1080, and 1813 mg/L.ConclusionIt is possible to reproducibly prepare highly purified calprotectin antigen from human granulocytes. The preparations can be used for preparing calibrators, controls for immunological calprotectin assays, and immunisation for raising antibodies against human calprotectin in hens.

Extraction, purification of prostate-specific antigen (PSA), and establishment of radioimmunoassay system as a diagnostic tool for prostate disorders

ABSTRACTThis study aimed to provide an easy and effective method for extraction and purification of prostate-specific antigen (PSA) from human seminal fluid with high quantity (14 mg) and high purity (98%). The obtained PSA was injected into rabbits for production of anti-PSA polyclonal antibody (titer 1/1000), labeled with radioactive iodine-125 for preparation of radioactive PSA tracer (purity 98 ± 1.8% and specific activity 64 ± 1.9 µCi/µg), and used in preparation of PSA standards. All prepared components can be used in PSA immunoassays specially radioimmunoassay (RIA) kit preparation as a diagnostic tool for prostatic diseases.

Isolation, Purification and Evaluation of Serological Activity of Rabies Virus Antigens

Objective of the study is to evaluate the serological activity of rabies virus antigens isolated from the brain tissue of mice by homogenization on FastPrep followed by ultracentrifugation. Materials and methods. Producer strain of the rabies virus “Ovechiy” GNKI. The rabies virus was isolated from the brain tissue of experimentally infected mice, followed by the study of the electrophoretic profile. The serological activity of the virus components was assessed by immunoblot and ELISA using specific anti-rabies sera. Results and conclusions. In the course of comparing the methods of isolation and purification of the rabies virus antigen, it was found that most optimal one is to use a homogenization on FastPrep-24, followed by fractionation in a sucrose gradient. As a result of fractionation in a graded sucrose density gradient with a concentration of 15–50 % at 25000 g for 120 min, five fractions of the rabies virus components were obtained. The maximum purified protein fraction was from 15–20 % sucrose zone, which corresponded to a molecular weight of 67 kDa. The specific antigen activity of the fraction in ELISA reached up the titers of 1:1280 (Specificity coefficient 2.2). Using immunoblot of antigens, obtained from the sucrose gradient in the range of 40–45 % and 20–35 % after ultracentrifugation, one major fraction of polypeptides (54 kDa) was detected, which showed the highest antigenic activity. The results obtained will be useful in the design of test systems for rabies screening and monitoring the effectiveness of anti-epizootic measures.

Development of an immunochromatographic strip for the rapid detection of maduramicin in chicken and egg samples

ABSTRACT Following the steps of antigen synthesis, immunization, cell fusion, ascites preparation and purification, a maduramicin (MD) monoclonal antibody (mAb) was produced. This MD-mAb demonstrated a 50% inhibition concentration value of 3.75 ng/ml, an affinity constant of 3.70 × 1010 l/mol, and the isotype was IgG3. The MD-mAb has no cross-reactivity with other polyether antibiotics. Using this MD-mAb, a gold immunochromatographic assay was developed to detect MD residues in chicken breast and egg samples. For semi-quantitative detection by the naked eye, the visual limit of detection was 5 ng/g in chicken breast, 10 ng/g in egg. Quantitative results can be obtained by a hand-held strip scan reader, with the detection range of 5.11–19.34 ng/g in chicken breast and 6.46–27.87 ng/g in egg. The strip test took 10 min to run in total. This strip assay is suitable for on-site detection of MD residues in chicken breast and egg samples.

Non-protease native allergens partially purified from bodies of eight domestic mites using p-aminobenzamidine ligand

BackgroundOptimised purification steps for concentrating trace target native antigens are needed. Combining the p-aminobenzamidine ligand with protease inactivation enables partial purification of mite non-protease allergens lacking proteases. ObjectiveWe sought to analyse in detail proteins obtained using this method from eight species of synanthropic acaridid mites and tested IgE reactivity using pooled human sera. Materials and methodsProteins affinity bound to p-aminobenzamidine as a ligand were identified by MALDI TOF/TOF. After electroblotting, the proteins were visualised using the fluorescent SYPRO-Ruby protein blot stain, and IgE reactivity was further analysed using pooled human sera collected from patients allergic to house dust mites. ResultsMS/MS identification confirmed previous results that no proteases were purified. Protein patterns corresponding to the allergens Der f 7, Der f 30 and actins indicated that these proteins are purified using p-aminobenzamidine and are present across a wide spectrum of acaridid mites. When using Dermatophagoides farinae, apolipophorins (Der f 14), chitinase-like Der f 15 and 18, 70-kDa heat shock protein, and a Der f Alt a10 allergen homolog (gi|37958173) were also detected. The target antigens tropomyosins and paramyosins showed similar IgE binding among the mite species tested. IgE reactivity with miscellaneous D. farinae antigen was also observed. ConclusionsPartial purification of mite non-protease antigens using a strategy combining p-aminobenzamidine with protease inactivation was verified by 1D-E and 2D-E analyses. IgE binding to p-aminobenzamidine-purified native non-protease mite antigens was tested using pooled sera. This preliminary study allows for further work on individual serum samples, allowing confirmation of immunoreactivity.

High-precision quantitation of a tuberculosis vaccine antigen with capillary-gel electrophoresis using an injection standard

Analysis of proteinogenic vaccine antigens in a quality control environment requires an accurate, precise, and reliable method for protein separation and quantitation. While having multiple advantages over the classical SDS-PAGE, capillary gel electrophoresis (CGE) has not yet become a standard tool in vaccine antigen analysis. Here we report on development of a CGE-based method for quantitative analysis of a tuberculosis vaccine fusion antigen protein, H4, currently in clinical trials. We demonstrate that our method can monitor antigen purity and relative quantity with greater precision and accuracy versus SDS-PAGE. In addition, due to use of direct light-absorbance detection, the CGE method is suitable for absolute quantitation, an application for which SDS-PAGE is limited due to the need for staining and limited dynamic range of detection. To further improve the performance of our quantitation method, we introduced Bovine Serum Albumin (BSA) as an injection standard to correct for signal variance associated with the injected sample volume. We found that, for our specific application, BSA was more appropriate as an injection standard versus one provided in a commercial kit, in terms of precision and accuracy for quantitation of H4. In addition to providing better method performance versus SDS-PAGE, CGE is also faster and less resource-intensive. We conclude that CGE should be considered as a replacement for traditional SDS-PAGE methods for vaccine antigen quantitation in a quality-control environment.

Development and Application of Synthetic Affinity Ligands for the Purification of Ferritin-Based Influenza Antigens.

A recently developed novel recombinant influenza antigen vaccine has shown great success in preclinical studies in ferrets and mice. It provides broader protection, and is efficient to manufacture compared to the conventional trivalent influenza vaccines (TIV). Each strain of the recombinant antigen has a constant self-assembled bacterial ferritin core which, if used as a target for affinity chromatography, could lead to a universal purification method. Ferritin in silico models were used to explore potential target binding sites against ligands synthesized by the four-component Ugi reaction. Two ligands, SJ047 and SJ055, were synthesized in solution, characterized by 1H, 13C, and 2D NMR spectroscopy, and subsequently immobilized on the PEG-functionalized beads. Ligands SJ047 and SJ055 displayed apparent Kd values of 2.04 × 10-7 M and 1.91 × 10-8 M, respectively, against the ferritin. SJ047 and SJ055-functionalized resins were able to purify hemagglutinin (New Caledonia)-ferritin expressed in a crude Human Embryonic Kidney (HEK) cell supernatant in a single step to a purity of 85 ± 0.5% (97 ± 1% yield) and 87.5 ± 0.5% (95.5 ± 1.5% yield), respectively. Additionally, SJ047 and SJ055-functionalized resins purified the recombinant antigens when spiked at known concentrations into HEK supernatants. All three strains, hemagglutinin (New Caledonia)-ferritin, hemagglutinin (California)-ferritin, and hemagglutinin (Singapore)-ferritin were purified, thereby offering an ideal alternate platform for affinity chromatography. Following elution from the affinity adsorbents, absorbance at 350 nm showed that there was no aggregation of the recombinant antigens and dynamic light scattering studies further confirmed the structural integrity of the recombinant antigen. The use of Ugi ligands coupled to a PEG-spacer arm to target the ferritin core of the strain is entirely novel and provides an efficient purification of these recombinant antigens. This approach represents a potentially universal method to purify any ferritin-based vaccine.

Immunometric Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay.

The double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) is preferentially used to determine the concentration of unknown antibody in a sample. Pure antigen is not required in this assay; however, the use of a reporter-labeled detection antibody is essential. The double-antibody sandwich ELISA is suitable for epitope mapping of different monoclonal antibodies that have been generated against a single antigen. First, plates are coated with a capture antibody specific for immunoglobulins generated by immunization of a host species. Next, the test antibody solution (e.g., serum) is incubated with the capture antibody to facilitate binding. The plates are washed to remove unbound antibody, and then antigen is added. The plates are washed again followed by the addition of an antigen-specific reporter-labeled antibody. Following incubation, unbound reporter antibody is washed off, and reporter-specific substrate is added. Reporter-mediated substrate hydrolysis is visualized and measured. The signal is proportional to the number of test antibodies present in the serum.

Isolation and purification of HLA-DR antigen from Daudi cell line by immunoaffinity chromatography

Isolation and purification of HLA-DR antigen from Daudi cell line by immunoaffinity chromatography

Process development for production and purification of the Schistosoma mansoni Sm14 antigen

The trematode Schistosoma mansoni Sm14 antigen was expressed in the yeast Pichia pastoris and secreted into the culture medium at yields of approximately 250 mg L−1. Sm14 belongs to a family of fatty-acid binding proteins and appears to play an important role in uptake, transport, and compartmentalization of lipids in S. mansoni and it is a potential vaccine candidate in both humans and domesticated animals. The Sm14 gene was codon-optimized for expression in P. pastoris, and placed under transcription of the strong methanol inducible AOX1 promoter. Mut+ transformants were selected and used in fed-batch cultivation using a 2.5L fermenter equipped with an on-line methanol control system in order to maintain constant methanol levels during induction. Optimal conditions for the expression of Sm14 by P. pastoris were found to be: dissolved oxygen at 40%, temperature of 25 °C, pH 5.0, and a constant methanol concentration of 1 gL-1. Our results show that a correctly processed Sm14 was secreted into the culture medium at levels of approximately 250 mg L−1. Sm14 from clarified culture medium was purified using a two-step procedure: anion-exchange chromatography followed by hydrophobic interaction chromatography, resulting in >95% purity with a final yield of 40% from the starting cell culture medium. This product has been tested in preliminary clinical trials and shown to elicit an antibody response with no adverse reactions.

A purified MAA-based ELISA is a useful tool for determining anti-MAA antibody titer with high sensitivity.

Atherosclerosis is widely accepted to be a chronic inflammatory disease, and the immunological response to the accumulation of LDL is believed to play a critical role in the development of this disease. 1,4-Dihydropyridine-type MAA-adducted LDL has been implicated in atherosclerosis. Here, we have demonstrated that pure MAA-modified residues can be chemically conjugated to large proteins without by-product contamination. Using this pure antigen, we established a purified MAA-ELISA, with which a marked increase in anti-MAA antibody titer was determined at a very early stage of atherosclerosis in 3-month ApoE-/- mice fed with a normal diet. Our methods of Nε-MAA-L-lysine purification and purified antigen-based ELISA will be easily applicable for biomarker-based detection of early stage atherosclerosis in patients, as well as for the development of an adduct-specific Liquid Chromatography/Mass Spectrometry-based quantification of physiological and pathological levels of MAA.

Immunological Principles Guiding the Rational Design of Particles for Vaccine Delivery.

Despite the immense public health successes of immunization over the past century, effective vaccines are still lacking for globally important pathogens such as human immunodeficiency virus, malaria, and tuberculosis. Exciting recent advances in immunology and biotechnology over the past few decades have facilitated a shift from empirical to rational vaccine design, opening possibilities for improved vaccines. Some of the most important advancements include (i) the purification of subunit antigens with high safety profiles, (ii) the identification of innate pattern recognition receptors (PRRs) and cognate agonists responsible for inducing immune responses, and (iii) developments in nano- and microparticle fabrication and characterization techniques. Advances in particle engineering now allow highly tunable physicochemical properties of particle-based vaccines, including composition, size, shape, surface characteristics, and degradability. Enhanced collaborative efforts between researchers in immunology and materials science are expected to rise to next-generation vaccines. This process will be significantly aided by a greater understanding of the immunological principles guiding vaccine antigenicity, immunogenicity, and efficacy. With specific emphasis on PRR-targeted adjuvants and particle physicochemical properties, this review aims to provide an overview of the current literature to guide and focus rational particle-based vaccine design efforts.

Physicochemical characterization and immunological properties of Pichia pastoris based HPV16L1 and 18L1 virus like particles

There continues to be an urgent need for cost-effective prophylaxis for HPV-associated cancers in socio-economically underdeveloped nations. Presently HPV vaccines, which are commercially available, are adjuvanted virus-like particles (VLPs) expressed from various recombinant expression systems. They have been characterized by different methods as safe, pure, and potent HPV vaccine antigens. We cloned and expressed L1 proteins of HPV16 & 18 in Pichia pastoris and tested their immunogenicity. We observed that HPVL1 proteins (16L1 and 18L1) are expressed in Pichia pastoris at high levels. Critical physicochemical parameters of these HPV recombinant L1 proteins were characterized by SDS PAGE, western blotting, peptide mapping, glycosylation pattern, mass spectrometry, host cell DNA and protein analysis, electron microscopy, and immunogenicity analysis. These data establish a blueprint of HPV recombinant protein antigens for standardizing & developing an alternative high-quality, cost-effective vaccine for HPV as well as similar recombinant protein-based vaccines.

Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule.

From 2013 to 2015, peste des petits ruminants (PPR) broke out in more than half of the provinces of China; thus, the application and development of diagnostic methods are very important for the control of PPR. Here, an immunoperoxidase monolayer assay (IPMA) was developed to detect antibodies against PPR. However, during IPMA development, we found that Vero cells were not the appropriate choice because staining results were not easily observed. Therefore, we first established a baby hamster kidney-goat signaling lymphocyte activation molecule (BHK-SLAM) cell line that could stably express goat SLAM for at least 20 generations. Compared with Vero cells, the PPR-mediated cytopathic effect occurred earlier in BHK-SLAM cells, and large syncytia appeared after virus infection. Based on this cell line and recombinant PPR virus expressing the green fluorescent protein (GFP) (rPPRV-GFP), an IPMA for PPR diagnosis was developed. One hundred and ninety-eight PPR serum samples from goats or sheep were tested by the IPMA and virus neutralization test (VNT). Compared with the VNT, the sensitivity and specificity of the IPMA were 91% and 100%, respectively, and the coincidence rate of the two methods was 95.5%. The IPMA assay could be completed in 4 h, compared with more than 6 d for the VNT using rPPRV-GFP, and it is easily performed, as the staining results can be observed under a microscope. Additionally, unlike the VNT, the IPMA does not require antigen purification, which will reduce its cost. In conclusion, the established IPMA will be an alternative method that replaces the VNT for detecting antibodies against PPRV in the field.

Construction of stabilized and tagged foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease that affects cloven-hoofed animals worldwide. Construction and purification of stable antigen for vaccine are necessary but technically difficult and laborious. Here, we have tried to investigate an alternative method by inserting a hexa-histidine tag (6xHIS) in the VP1 C-terminal for easy purification and replacing two amino acids of VP1/VP2 to enhance the stability of the capsid of the FMD virus (FMDV) Asia1/MOG/05. In addition, infectious 6xHIS-tagged stable (S/T) FMDVs were maintained under acidic conditions (pH 6.0) and were readily purified from small-scale cultures using a commercial metal-affinity column. The groups vaccinated with the S/T FMDV antigen showed complete protection comparing to low survival rate in the group vaccinated with non-S/T FMDV against lethal challenge with Asia1 Shamir in mice. Therefore, the present findings indicate that the stabilized and tagged antigen offers an alternative to using the current methods for antigen purification and enhancement of stability and has potential for the development of a new FMD vaccine.

P120 Correlation between FlowPRA® and LABScreen Class II single antigen beads for the detection of HLA DQB antibodies: Not all assays are created equal

Aim Several publications have shown the clinical importance of donor-specific DQ antibodies, post-transplant, in kidney, heart and lung. We wanted to determine if FlowPRA®(F-PRA) screening beads would be a cost-effective way to assess the presence of DQB antibodies. Methods Patients with monospecific and well defined DQB specificities were identified for testing. Both F-PRA screen and LABScreen Class II single antigen bead (SAB) (One Lambda, Inc) assays were performed using serial dilutions of these sera to determine the detection “end-point” for each method. Results Our data showed that for DQB1 specificities, ∗02, ∗04, ∗05/∗06, and ∗03:01/02/03, when MFI values were high; eg: >10,000, the F-PRA displayed distinct positive staining architecture. However, when diluted, SABs could identify the presence of DQB antibody at dilutions that were negative by F-PRA (Fig. 1). The corresponding SAB MFI values ranged from ∼ 750 – 5300 (Table 1). Conclusions Our data show that the F-PRA will fail to detect HLA DQB antibody at concentrations that yield moderate MFI values by LABScreen Class II SABs. Hence, F-PRA cannot be used alone to rule out the presence of HLA DQB antibodies. The reason for this discrepancy may be differences in antigen density between the two products as each one uses a different type of cell line for antigen purification. We are in the process of performing similar studies, including crossmatches, for other HLA loci. Download : Download high-res image (220KB) Download : Download full-size image Download : Download high-res image (66KB) Download : Download full-size image

Purification of 146s Antigen of Foot-and-Mouth Disease (FMD) Virus Serotypes Aby Using the Sucrose Gradient Procedure

Background: Foot-and-Mouth disease (FMD) is an acute and contagious disease in domestic ruminants, which is currently the most economical viral disease that threatens the livestock industry. The virus that causes disease belongs to the Aphthovirus genus from the family of picornaviridae. This family contains seven serotypes and is about 30 nanometers in diameter and has no external membrane, similar to other picornaviruses. Objectives: In this study, we aimed to introduce the sucrose gradient procedure as a convenient method for purification of 146s antigens. Methods: Sucrose gradient procedure (20% - 50%) was used for purification of 146s antigen of foot-and-mouth disease virus serotypes A. Then, preparation steps of the virus including concentration by polyethylene glycol, degreasing using trichloroethy- lene, centrifugation (30000 g for three hours) and washing the pellet using Tris (0.05 M) were performed. Spectrophotometer and nano-drop were used to measure the amount of the purified protein and purity evaluation, respectively. Moreover, dot blot assay was used for the confirmation of 146s antigen. Results: The pellet of the 146s antigen of FMD virus serotype A was developed at a concentration of sucrose 50%. Absorbance rate of the Foot-and-Mouth Disease virus serotypes A at wavelengths of 240, 259 and 280 nm was 1.238, 1.573 and 1.157, respectively. Moreover, the amount of 146s antigen at the same wavelengths was 163.416, 207.636 and 152.724 g/mL, respectively. The amount of purified protein by nanodrop (ND-1000, the United States) was 0.275 mg/mL. The 146s antigen was observed with 26, 29 and 64 KDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and was confirmed by the dot blot assay. Conclusions: The results exhibited that sucrose gradient procedure is a good method for purification of virus.

Purification of the Specific Antibody of Foot-and-Mouth Disease Virus Serotype A by Ion Exchange Chromatography

Background: Foot-and-mouth disease (FMD) is an acute and contagious disease in domestic ruminants, which is currently the most economical viral disease that threatens livestock industry. The virus that causes the disease is belongs to Aphthovirus genus from the picornaviridae family. This family contains seven serotypes and is about 30 nanometers in diameter and no external membrane similar to other picornaviruses. Objectives: The current study aimed to introduce ion exchange chromatography as a convenient method to purify specific antibody against 146S antigen. Methods: The study was performed in vaccine and serum research of Razi institute, Tehran, Iran, after purification of 146S antigen of foot-and-mouth disease virus serotypes A using sucrose gradient procedure; two Guinea pigs were immunized with 30 µg 146S antigen combined with complete Freund’s adjuvant (CFA) and booster with incomplete Freund’s adjuvant (IFA) according to the protocol. After bleeding and serum obtaining, agglutination assay, dot blot and the enzyme linked immunosorbent assay (ELISA) were used to confirm specific antibody against 146S. Ion exchange chromatography was used to purify specific antibody against 146S antigen. Finally, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis was used to evaluate the purification. Results: The interaction of 146S antigen with the immunized rabbit resulted in agglutination reaction. Assaying with the heterologous antigen showed negative result that confirmed the production of specific antibody in the rabbit. In dot blot, presence of brown spots was confirmed by binding specific antibody with 146S antigen. The result of ELISA showed that each antigen of different serotypes reacted better with homolog antibody. Since IgG containing positive charge and 25, 2-diethylaminoethyl (DEAE) Cephadex gel in ion exchange chromatography also had positive charge, IgG was eluted at the first step by buffer (pH = 9) and the other proteins were eluted by buffer at different pH levels. The absorbance amount of IgG was 0.197 that showed IgG = 0.725 mg/mL. Conclusions: With respect to the results as well as the speed of ion exchange chromatography, this method is advised to purify antibodies.

Isolation and purification of Echinococcus granulosus antigen B from hydatid cyst fluid using three different methods.

Hydatid cyst, the larval stage of cestodes Echinococcus spp., is recognized as a zoonotic infection in the world. The World Health Organization (WHO) has recently classified echinococcosis in a group of neglected tropical diseases. The prevalence of Echinococcus granulosus infection is high in Iran due to the presence of various intermediate hosts in this country. Considering the rising trend of this zoonotic parasitic disease based on national epidemiological studies, diagnosis is of great significance. WHO has suggested the use of specific antigens, especially antigen B (AgB) for serological diagnostic tests. In general, AgB is a polymeric lipoprotein, which disintegrates into 8.12, 16, and 20.24 kDa subunits. In the present study, we applied three different methods for AgB isolation from hydatid cyst fluid (HCF) and compared their efficacy in AgB isolation. Finally, the protein concentration of this antigen was measured by Bradford assay and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that the application of polyethylene glycol (PEG 4000) as a thickener agent beside purification of HCF in dialysis bag and filtering and also dialysis against acetate buffer leading to the best quantity in purified antigen B.

Use of immunoblotting in testing Madurella mycetomatis specific antigen.

Though serodiagnosis of actinomycetoma is established, that of eumycetoma due to Madurella mycetomatis is limited because of lack of pure antigen. Reliable rapid tests are needed to make an accurate timely diagnosis. The purpose of this study is to detect antigen parts of M. mycetomatis, which act specifically with M. mycetomatis antibodies. Cytoplasmic antigen was prepared from molecularly identified cultures of M. mycetomatis by sonication, ultracentrifugation, dried, weighed and appropriately reconstituted. M. mycetomatis cytoplasmic antigen were separated using 12% sodium dodecyl sulfate-polyacrylamide gel, and immunoblotting to detect the reactive ones.Immunoblotting was carried out in nitrocellulose strips containing different molecular size. Sera from patients and co-patients as control were used. When stained with Coomassie brilliant blue R 250 seven molecular weights appeared but only three, 45, 60, 95 kDa reacted with M. mycetomatis patients few from control group, one from a malaria patient. No reactive band was observed with sera from actinomycetoma, Aspergillus flavus-associated aspergillosis, schistosomiasis, leishmaniasis, fungal sinusitis nor healthy controls. Specific fractions of M. mycetomatis antigen which were demonstrated by immunoblotting showed 75% sensitivity and 95% specificity. The true negative tests were 14 patients (32.5%). This also means that immunoblotting is reasonably reliable in diagnosis and follow-up of eumycetoma patients.